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RNA processing in eukaryotes

Published byClifton Young Modified over 8 years ago

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Presentation on theme: "RNA processing in eukaryotes"— Presentation transcript:

1 RNA processing in eukaryotes
DNA promoter exons introns primary transcript (nucleus) 5’ cap AAAAAAAAA 3’ poly - A tail splicing transcription unbroken coding sequence transport to cytoplasm for translation final mRNA

2 5′ cap methylated guanine “backward” 5′ to 5′ linkage
Not encoded in DNA Capping enzyme Recognition by ribosome 5′ AGACCUGACCAUACC

3 RNA processing in eukaryotes
DNA promoter exons introns primary transcript (nucleus) 5’ cap AAAAAAAAA 3’ poly - A tail splicing transcription unbroken coding sequence transport to cytoplasm for translation final mRNA

4 3′ poly(A) tail Poly(A) polymerase Add ~200 A’s Not in template
Important for: Export of mRNA Initiation of Translation Stability of mRNA …UGGCAGACCUGACCA 3′ …UGGCAGACCUGACCAAAAAAAAAAAAAAAAAAAA

5 RNA processing in eukaryotes
DNA promoter exons introns primary transcript (nucleus) 5’ cap AAAAAAAAA 3’ poly - A tail splicing transcription unbroken coding sequence transport to cytoplasm for translation final mRNA

6 Splicing Most genes interrupted by introns
Introns removed after transcription Exons spliced together 5’ cap AAAAAAAAA 3’ poly - A tail splicing splicing AAAAAAAAA final mRNA unbroken coding sequence

7 Splicing snRNPs recognize exon-intron boundaries RNA + protein
Cut and rejoin mRNA

8 Splicing RPE65 mRNA in nucleus: 21,000 nt (14 exons) AAAAAAAAA
mature RPE65 mRNA in nucleus: 1,700 nt (8%)

9 Splicing Alternative splicing: >1 protein from one gene
27,000 human genes, but >100,000 proteins

10 Splicing Mutations affecting splicing can cause genetic disease:
cystic fibrosis retinitis pigmentosa spinal muscular atrophy Prader-Willi syndrome Huntington disease spinocerebellar ataxia myotonic dystrophy Fragile-X syndrome Or produce genetic susceptibility to disease: lupus bipolar disorder schizophrenia myocardial infarction type I diabetes asthma cardiac hypertrophy multiple sclerosis autoimmune diseases elevated cholesterol

11 Gene expression summary
Prokaryotes Eukaryotes DNA DNA transcription transcription mRNA pre-mRNA cytoplasm nucleus capping polyadenylation splicing directly translated (even before being completely transcribed) protein mature mRNA transport to cytoplasm translation cytoplasm protein

12 Quick review of protein structure
amino acids generic amino acid

13 Quick review of protein structure
side chain gives chemical properties Non-polar (hydrophobic): Charged: Polar, not charged: Negative: Positive:

14 Quick review of protein structure
polymer of amino acids = polypeptide ≈ protein methionine aspartate

15 Quick review of protein structure
polymer of amino acids = polypeptide ≈ protein N- terminus C- terminus peptide bond methionine aspartate

16 Quick review of protein structure
polymer of amino acids = polypeptide ≈ protein methionine aspartate glycine

17 Quick review of protein structure
polymer of amino acids = polypeptide ≈ protein methionine aspartate glycine phenylalanine

18 Quick review of protein structure
polymer of amino acids = polypeptide ≈ protein methionine aspartate glycine phenylalanine valine

19 Quick review of protein structure
polymer of amino acids = polypeptide ≈ protein methionine aspartate glycine phenylalanine valine lysine

20 Quick review of protein structure
What holds folded proteins together? Hydrogen bonds Hydrophobic interactions Ionic bonds Disulfide bonds (covalent) …all determined by amino-acid sequence

21 Quick review of protein structure
Primary (1°) structure Secondary (2°) structure alpha helix Tertiary (3°) structure beta sheet Quaternary (4°) structure hemoglobin L-isoaspartyl protein carboxyl methyltransferase

22 Translation Ribosome finds start codon within mRNA
Genetic code determines amino acids Stop codon terminates translation start codon stop codon mRNA 5′ coding region 3′ 5′ UTR 3′ UTR translation protein NH3 COOH

23 Ribosome Large ribonucleoprotein structure
E. coli: 3 rRNAs, 52 proteins Two subunits: large and small large subunit RNA small subunit protein

24 Eukaryotic Translation
How does the ribosome find the correct start codon? Small ribosome subunit binds 5′ cap Scans to first AUG start codon stop codon cap mRNA 5′ coding region AAAAAAAAA… 3′ 5′ UTR 3′ UTR

25 Prokaryotic Translation
How does the ribosome find the correct start codon? Small subunit binds Shine-Dalgarno sequence (RBS) Positioned correctly for translation start codon stop codon mRNA 5′ Shine-Dalgarno sequence or RBS (AGGAGG) coding region 3′ 5′ UTR 3′ UTR

26 the Genetic Code After finding start codon, use the genetic code:
Shown as mRNA 5′ → 3′ 26

27 Mechanics of Translation
Translation requires: mature mRNA ribosome tRNAs amino acids accessory proteins

28 tRNA Small RNAs (74-95 nt) made by transcription
Intramolecular base pairing Anticodon complementary to mRNA codon anticodon

29 tRNA “Charged” by specific aminoacyl tRNA synthetase

30 Initiation of Translation
Small ribosome subunit binds at start codon Prokaryotes: Shine-Dalgarno sequence (RBS) Eukaryotes: binds cap, scans mRNA 5′ AUG GAU GGG

31 Initiation of Translation
First tRNA (Met, anticodon CAU) joins complex 5' 3' Met UAC mRNA 5′ AUG GAU GGG

32 Initiation of Translation
Large ribosomal subunit joins 5' 3' Met UAC mRNA 5′ AUG GAU GGG

33 Initiation of Translation
P site holds tRNA with first aa A site open for next tRNA P A 5' 3' Met UAC mRNA 5′ AUG GAU GGG

34 Initiation of Translation

35 Elongation Next tRNA enters CUA P A UAC mRNA 5′ AUG GAU GGG Asp 3' 5'
Met UAC mRNA 5′ AUG GAU GGG

36 Elongation Peptidyl transferase forms peptide bond
Amino acid released from tRNA in P site Met Met Asp mRNA 5′ UAC 3' 5' CUA 3' 5' AUG GAU GGG

37 Elongation Ribosome translocates one codon
First tRNA binds briefly in E site until translocation completes Met Asp 5' 3' UAC mRNA 5′ CUA 3' 5' AUG GAU GGG

38 Elongation Process repeats Next tRNA can then enter the empty A site
5' 3' Gly CCC Met Asp P A mRNA 5′ CUA 3' 5' AUG GAU GGG

39 Elongation

40 Termination Ribosome stops at stop codon No matching tRNA
Release factor binds Met Val Leu Asp Gly Phe Val Lys Gly Leu Gln P A Asp Ile RF GUC 3' 5' UUG CAG UAG

41 Termination Translation complex dissociates UUG CAG UAG GUC RF Met Val
Leu Asp Gly Phe Val Lys Gly Asp Leu Gln Ile UUG CAG UAG 5' 3' GUC RF

42 Polyribosomes Next ribosome starts as soon as start codon is available
Growing polypeptide N RNA subunits released Ribosome 3' 5' AUG mRNA Stop 5' – 3' direction of ribosome movement C N Released polypeptide

43 Operons More than one gene on one mRNA Prokaryotes only

44 Operons More than one gene on one mRNA Prokaryotes only

45 Protein Synthesis Pathways
Free ribosomes Ribosomes bound to RER

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